JP7396997B2 - Complete flow-through method for purifying recombinant proteins - Google Patents

Description

The present invention relates to a complete flow-through purification method for small and large scale purification of proteins, particularly monoclonal antibodies.

Antibody purification can be one of the most costly aspects of biological production. Monoclonal antibodies (mAbs) are generally purified using a three-step, three-resin chromatography method using at least one specific buffer system in each step. This conventional purification process involves an intermediate purification step after the capture step, completed with a polishing step, and typically takes 3 to 5 working days (including storage and open phases). In such conventional methods, these three steps are performed in a series of separate unit operations, which are operated in a continuous manner, as adjustments in pH, molarity, and protein concentration are required between each step. Can not do it. Therefore, conventional purification methods generally require a wide variety of buffers and multiple storage devices during each step stop and between several systems. Therefore, these conventional purification methods are prone to contamination, technical failures, and human error. In addition, it requires interruptions between each step for concentration of eluate, adjustment of pH and conductivity, and storage of eluate before the next step, and also before completion of the previous step. Due to the inability to start the process, such conventional purification methods are particularly long and costly.

Also, conventional methods are expensive because they typically use a Protein A matrix as the first purification step. In fact, historically, the most selective resins have generally been introduced early in the process for Protein A to remove as many impurities as possible. However, even though Protein A is the most selective medium for monoclonal antibody purification, there are still contaminants that remain in the process. This step is also the most expensive of all the methods since it is also used as the first step which comes into contact with large amounts of contaminants. In addition, residual contaminants are very difficult to remove to comply with pharmaceutical specifications.

As cell culture titers and cell culture volumes used for production increase, downstream processing becomes considered an industrial bottleneck. This is particularly important for monoclonal antibody production, where the focus has shifted from batch volumes to downstream processing volumes. Additionally, early preclinical and clinical phase studies require large amounts of antibodies that can be produced more rapidly. Therefore, for protein purification, in particular for antibody purification, and also for the reduction of both the time taken to obtain a batch and the requirement for scaling up the method at the risk of contamination, technical failure and human error. There is a need in industry for a less expensive process that can be carried out in a continuous manner.

The inventors have proposed a new method for purifying proteins, in particular antibodies, that involves only three steps in a continuous complete flow-through manner, none of these steps contain protein A, and one We have now found a method which advantageously uses only a small amount of buffer and still makes it possible to obtain high yields of purified antibodies with excellent purity. Purified proteins are therefore suitable for medical applications. As a result, this method can be used to purify proteins for clinical trials and/or for the manufacture of pharmaceutical compositions containing the proteins. In addition, the method does not require any intermediate step adjustments and thus can be carried out in a closed system from the harvest of protein to be purified to the final product.

Briefly, this method consists of only three consecutive steps, the first being an inexpensive technique aimed at removing a large amount of contaminants, the remaining small amount of contaminants being removed, and this Therefore, it includes techniques for use in the second and third steps, which are increasingly selective for use on a small scale compared to their use in conventional methods.

Thus, the method of the invention continuously comprises one filtration step comprising the use of at least one chelating agent, an exchange step comprising the use of at least one diafiltration membrane, and a polishing step comprising the use of a combination of membrane adsorbents. in which the two membrane adsorbents of said membrane adsorbent combination are orthogonal with respect to their mode of action. The method of the invention is schematically illustrated in FIG. These three purification steps are advantageously carried out in this particular order. In addition, it was found that only one buffer, the one used for the exchange step, needed to be used throughout the entire method. In other words, the equilibration buffer optionally used in the polishing step is advantageously the same as the buffer used in the exchange step. This buffer advantageously comprises Bis-Tris, Tris, Tris-HCl, phosphoric acid and/or citric acid in combination with, for example, NaCl, acetic acid and water. More specifically, only one buffer is used for the entire method, ensuring compatibility between all steps, allowing for supply chain, manufacturing and quality control savings, and reduced storage requirements. I can do it.

The method of the invention is an open-phase (i.e., a process in which the purification system is open for manual operations such as preparing the chromatography column for fresh buffer, diluting the sample, or adjusting its pH). It also allows for decommissioning, thereby reducing the risk of contamination and giving the possibility of working in a more undifferentiated environment. Additionally, because the method of the present invention advantageously does not involve the use of any columns, and primarily uses disposable, ready-to-use membrane adsorbents, there is no need for storage or confirmation of reuse, and no need for column preparation or packing. There are no associated controls or cleaning checks and equipment limitations. Accordingly, process cycle times are shortened, process scale-up requirements are minimized, and operating and storage costs can be reduced. Thus, the method of the invention allows for both rapid and cost-effective batch production and reduced purification system occupation time. Therefore, the method of the invention is suitable for scale-up and purification of recombinant proteins from bench to industrial scale.

A specific protocol was set up and executed for two different antibodies. In this protocol, the filtered protein solution obtained at the end of the first filtration step is passed directly through at least one diafiltration membrane without undergoing any treatment such as pH adjustment, buffer exchange or dilution. The residual liquid obtained at the end of the exchange step is passed directly through the combination of at least two membrane adsorbents without undergoing any treatment such as pH adjustment, buffer exchange or dilution. This protocol has the advantage of being very rapid (a few hours), yielding high yields (>70%), purity comparable to pharmaceutical industry standards, and allowing for a reduction in both buffers and storage equipment used. do. Moreover, this method is very flexible and has cost-saving advantages, since it does not involve the use of a protein A matrix, which is generally the most expensive element of conventional methods. In addition, the process can be carried out in a fully automated and continuous manner and does not involve any open phase. Moreover, this method was successfully performed on two different antibodies.

Accordingly, the present invention provides a method for purifying proteins from solution, comprising:
(a) a filtration step,
- passing said solution through at least one chelating agent matrix in a flow-through manner;
- recovering a filtered protein solution from the flow-through of said at least one chelating agent matrix;
(b) a replacement step,
- passing the filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for exchange;
- recovering a retentate of said at least one diafiltration membrane comprising partially purified protein;
(c) a polishing step,
- passing the residual liquid obtained at the end of step (b) in a flow-through manner through a combination of membrane adsorbents, the two membrane adsorbents of said combination of membrane adsorbents having a mechanism of action; and the combination of membrane adsorbents is pre-equilibrated with an equilibration buffer identical to the one buffer used for exchange in step (b);
- recovering purified protein from the flow-through of said membrane adsorbent combination, and does not include a chromatography step with Protein A.

The present invention also provides a method for purifying proteins from a solution, comprising:
(a) a filtration step,
- passing the solution through at least one chelating agent matrix in a flow-through manner;
- recovering a filtered protein solution from the flow-through of said at least one chelating agent matrix;
(b) a replacement step,
- passing the filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for exchange, said only one the buffer of is the same as the equilibration buffer used in polishing step (c);
- recovering a retentate of said at least one diafiltration membrane comprising partially purified protein;
(c) a polishing step,
- passing the residual liquid obtained at the end of step (b) in a flow-through manner through a combination of membrane adsorbents, the two membrane adsorbents of said combination of membrane adsorbents having a mechanism of action; and the combination of membrane adsorbents is pre-equilibrated with an equilibration buffer;
- recovering purified protein from the flow-through of said membrane adsorbent combination, and does not include a chromatography step with Protein A.

The invention particularly relates to a method for purifying proteins from solution, comprising:
(a) a filtration step,
(i) passing the solution through the at least one chelating agent matrix in a flow-through manner;
(ii) recovering a filtered protein solution from the flow-through of the at least one chelating agent matrix;
(b) a replacement step,
(i) passing the filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for exchange;
(ii) recovering a retentate of the at least one diafiltration membrane comprising partially purified protein; and (c) a polishing step, the step comprising:
(i) passing an equilibration buffer through the combination of membrane adsorbents, said equilibration buffer being the same as one buffer used for exchange in step (b);
(ii) passing the residual liquid obtained from step (b) through the membrane adsorbent combination in a flow-through manner;
(iii) recovering purified protein from the flow-through of the membrane adsorbent combination, and does not include a Protein A chromatography step.

In one embodiment of the invention, only one buffer is used throughout the purification process. In certain embodiments, one buffer comprises Tris, Tris-HCl, Bis-Tris, phosphoric acid and/or citric acid. In another embodiment, one buffer comprises or consists of (i) Bis-Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) water, and (iv) optionally a salt.

In one embodiment, at least one chelating agent matrix of the filtration step is selected from activated carbon, diatomaceous earth, free cation exchange resins, free anion exchange resins, and free mixed mode resins. In certain embodiments, the at least one chelating agent matrix is a combination of two different chelating agent matrices. In a more specific embodiment, the combination of two different chelating agent matrices is a combination of activated carbon and free anion exchange resin. In another specific embodiment, the at least one chelating agent matrix is a combination of three or more, ie, three, four, five or six or more different chelating agent matrices.

In one embodiment, at least one diafiltration membrane of the exchange step is a single path tangential flow filtration (SPTFF) module or a tangential flow filtration (TFF) module. In certain embodiments, at least one diafiltration membrane of the exchange step is in the form of a cassette, hollow fiber or spiral. In certain embodiments, the filtered protein solution is concentrated during the exchange step.

In one embodiment, the membrane adsorbent combination of the polishing step is a combination of two membrane adsorbents, or a combination of at least two membrane adsorbents, such as three, four or at least four membrane adsorbents.

In one embodiment, the membrane sorbent of the membrane sorbent combination consists of a cation exchange membrane sorbent, an anion exchange membrane sorbent, a multimodal membrane sorbent, a hydrophobic interaction membrane sorbent, and combinations thereof. selected from the group. In certain embodiments, the membrane adsorbent combination of the polishing step is a combination of a cation exchange membrane adsorbent and an anion exchange membrane adsorbent.

In one embodiment, the combination of membrane adsorbents for the polishing step is at least selected from the group consisting of cation exchange membrane adsorbents, anion exchange membrane adsorbents, multimodal membrane adsorbents, and hydrophobic interaction membrane adsorbents. It is a combination of two membrane adsorbents.

In one embodiment of the invention, the method further comprises a nanofiltration step after step (c) and optionally a final ultrafiltration and/or diafiltration step after the nanofiltration step. In another embodiment of the invention, the method further comprises a low pH inactivation step after step (c), after the nanofiltration step, and/or after the final ultrafiltration and/or diafiltration step. . In one embodiment of the invention, the method comprises, before step (a), the step of culturing the cells in a liquid culture medium, preferably in a bioreactor, providing a liquid culture medium comprising the protein. The cultured cells can be mammalian, bacterial or yeast cells. In a preferred embodiment, the cultured cells are mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, HEK293 cells, etc., including various subtypes of these cell lines) as well as primary or established cells. or mammalian cell culture (eg, produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neural cells, adipocytes, etc.).

In one embodiment of the invention, the protein to be purified is an antibody. In another embodiment, the antibody is a monoclonal antibody.

Accordingly, the present invention also provides an integrated method for the production of purified proteins from liquid culture media.

In certain embodiments of the invention, one buffer comprises 5-40mM (especially 20mM) Bis-Tris, 15-150mM (especially 7.5), adjusted to a pH of 6-9 (especially 7.5) with acetic acid. 75mM) of NaCl.

In certain embodiments of the invention, one buffer comprises 5-40 mM (especially 20 mM) Tris or Tris-HCl, 15-40 mM (especially 20 mM), adjusted to a pH of 6-9 (especially 7.5) with acetic acid. Contains 150mM (specifically 75mM) NaCl.

In certain embodiments of the invention, one buffer comprises 5-40 mM (especially 20 mM) Tris or Tris-HCl, 15 Contains ~150mM (specifically 75mM) NaCl.

In certain embodiments of _the invention _{, one} buffer is _Na2HPO4 / _NaH2PO4 (preferably 10mM/90mM _Na2HPO4 / _NaH2PO4 to 90mM/10mM _{Na2HPO4 ) .} /NaH ₂ PO up to ₄ ) to a pH of 6-9 (especially 7.5).

The present invention provides a combination of at least one chelating agent matrix, at least one diafiltration membrane, and a membrane adsorbent, wherein the two membrane adsorbents of said membrane adsorbent combination are orthogonal with respect to their mechanism of action. , a combination of membrane adsorbents and Tris, Tris-HCl, Bis-Tris, phosphoric acid and/or citric acid, in particular (i) Bis-Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) water, and (iv) providing a kit comprising one buffer, optionally comprising or consisting of NaCl; In some embodiments, the kit is used to purify proteins from solution using the methods of the invention.

The present invention also provides a combination of at least one chelating agent matrix, at least one diafiltration membrane, and a membrane adsorbent, wherein two membrane adsorbents of said membrane adsorbent combination are orthogonal with respect to their mechanism of action. and a combination of membrane adsorbents which are: and Tris, Tris-HCl, Bis-Tris, phosphoric acid and/or citric acid, in particular (i) Bis-Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) A kit is provided that includes instructions for preparing a buffer comprising or consisting of water and (iv) optionally NaCl. In some embodiments, the kit is used to purify proteins from solution using the methods of the invention.

Also provided herein are isolated proteins, medicaments, and pharmaceutical compositions obtained by any of the methods described herein.

These and other features and advantages of the purification methods of the present disclosure will be more fully understood from the following detailed description, summarized by the appended claims. It is noted that the claims are defined by the detailed description of the features and advantages described herein, rather than by specific consideration of them.

In the context of the present invention, the terms "comprising", "having", "including" and "containing", unless stated otherwise, are used as non-limiting terms (i.e. "including"). ``but not limited to''). In addition, the term "comprising" includes "consisting of" (e.g., a composition "comprising" X can consist exclusively of X or can include something additional, such as X+Y) .

To simplify the protein purification process and make it inexpensive, the inventors developed a new purification process that is continuous, complete flow-through, and does not include a Protein A chromatography step.

The present invention is a method for purifying proteins from a solution, comprising:
(a) a filtration step,
- passing the solution through at least one chelating agent matrix in a flow-through manner;
- recovering a filtered protein solution from the flow-through of said at least one chelating agent matrix;
(b) a replacement step,
- passing the filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for exchange;
- recovering a retentate of said at least one diafiltration membrane comprising partially purified protein;
(c) a polishing step,
- passing the residual liquid obtained at the end of step (b) in a flow-through manner through a combination of membrane adsorbents, the two membrane adsorbents of said combination of membrane adsorbents having a mechanism of action; and the combination of membrane adsorbents is pre-equilibrated with an equilibration buffer identical to the one buffer used for exchange in step (b);
- Recovering purified protein from the flow-through of said membrane adsorbent combination, and not including a chromatography step with Protein A.

In exchange step (b), the expression "passing the filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for exchange" means passing said filtered protein solution and one buffer through at least one diafiltration membrane.

In certain embodiments, the method of the invention comprises:
(a) a filtration step,
(i) passing the solution through the at least one chelating agent matrix in a flow-through manner;
(ii) recovering a filtered protein solution from the flow-through of the at least one chelating agent matrix;
(b) a replacement step,
(i) passing the filtered protein solution obtained from step (a) through at least one diafiltration membrane using only one buffer;
(ii) recovering a retentate of the at least one diafiltration membrane comprising partially purified protein; and (c) a polishing step, the step comprising:
(i) passing an equilibration buffer through the combination of membrane adsorbents, said equilibration buffer being the same as one buffer used for exchange in step (b);
(ii) passing the residual liquid obtained from step (b) through the membrane adsorbent combination in a flow-through manner;
(iii) recovering purified protein from the flow-through of said membrane adsorbent combination, said purification method not including a Protein A chromatography step.

As shown above, the above method of the invention comprises only three steps, none of which are Protein A chromatography steps. Even if the method according to the invention comprises only three steps and does not include a chromatography step with Protein A, it is possible to obtain purified proteins suitable for pharmaceutical purposes and in particular for administration to humans.

In addition to the absence of human handling in the purification method (and the consequent reduction in the overall time required to complete the purification method), the disclosed method reduces the amount of buffer used for purification and reduces the amount of protein A The absence of a chromatography step reduces costs. The disclosed purification method also simplifies mAb purification, increases overall yield, reduces raw materials, storage equipment, product costs and processing time, while allowing purification of a wide variety of mAbs.

In contrast to conventional protein purification methods, as mentioned above, the method disclosed herein uses a unique buffer, which is used in both the exchange step and the membrane adsorbent in the polishing step. used for equilibration.

As used herein, "buffer according to the invention" refers to a buffer containing Bis-Tris, Tris, Tris-HCl, phosphoric acid and/or citric acid. Bis-Tris, Tris or Tris-HCl are compounds well known to those skilled in the art;
- The IUPAC name for Bistris is 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol, and the CAS number is 6976-37-0;
- Tris is 2-amino-2-(hydroxymethyl)propane-1,3-diol, CAS number 77-86-1;
- Tris-HCl is 2-amino-2-(hydroxymethyl)propane-1,3-diol hydrochloride, CAS number 1185-53-1.

Such buffers according to the invention may correspond to exchange buffers and equilibration buffers.

More particularly, such buffers according to the invention contain varying concentrations of the same chemicals, one of which is Bis-Tris, Tris, Tris-HCl, phosphoric acid and/or citric acid. Or it could become. In certain embodiments, the buffer comprises Bis-Tris, Tris, Tris-HCl, phosphoric acid and/or citric acid. In certain embodiments, the buffer comprises or consists of (i) Bis-Tris, Tris, or Tris-HCl, (ii) acetic acid, and (iii) water. In a more particular embodiment, the buffer comprises or consists of (i) Bis-Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) NaCl and (iv) water. In other cases, such buffers contain or consist of varying concentrations of (i) Bis-Tris, Tris, or Tris-HCl, (ii) acetic acid, (iii) NaCl, and (iv) water.

The exchange buffer comprises or consists of, for example, 5-40mM (e.g. 20mM) Bistris and 15-150mM (e.g. 75mM) NaCl, adjusted to a pH of 6-9 (e.g. 7.5) with acetic acid. obtain.

The exchange buffer may alternatively contain 5-40mM (especially 20mM) Tris or Tris-HCl, 15-150mM (especially 75mM) NaCl, adjusted to a pH of 6-9 (especially 7.5) with acetic acid. may contain or consist of these.

The exchange buffer can alternatively be 5-40mM (especially 20mM) Tris or Tris-HCl, 15-150mM (especially 75mM) NaCl, adjusted to a pH of 6-9 (especially 7.5) with citric acid. may contain or consist of these.

_The exchange buffer may alternatively be 6 _{% Na2HPO4} / _NaH2PO4 (preferably _from 10mM/90mM _{Na2HPO4 / NaH2PO4} to 90mM/ _{10mM Na2HPO4} / _NaH2PO4 ) _. It may contain or consist of 15-150mM (especially 75mM) NaCl adjusted to a pH of ~9 (especially 7.5).

Such exchange buffers are particularly suitable for use in exchange steps, in particular with TFF cassettes, but also in the equilibration of membrane adsorbents, particularly in combinations of cation exchange membrane adsorbents and anion exchange membrane adsorbents. It is also suitable for equilibration.

The advantage of the formulation of the buffer described above is that it passes the three steps of the method, in particular the exchange step and the polishing step, with even higher compatibility and, moreover, minimizes undesired interactions and reduces pH and conductivity. including the ability of the mAb product to limit the amount of oxidation and promote increased yield relative to traditional purification methods. The use of such a buffer formulation allows the process to be carried out without the need for any intermediate storage between the three steps (a), (b) and (c).

Consequently, in certain embodiments, the method does not include any intermediate preservation between the three steps (a), (b) and (c).

A sanitizing buffer may optionally be used when reusing the membrane adsorbent from the polishing process. Such a sanitizing buffer may comprise or consist of at least NaOH, more preferably 0.05N to 1N (eg 0.5N) NaOH.

The term "polypeptide" or "protein" as used herein refers to
1) a molecule having the sequence of a native protein, which is a) a protein produced by a naturally occurring, in particular non-recombinant, cell, or b) a genetically modified or recombinant cell, or 2) a Refers to a molecule that differs from the sequence of the native protein by deletions, additions, and/or substitutions of or more amino acids, and/or by at least one post-translational modification (eg, glycosylation).

The molecules mentioned in section 1) above may be referred to as native proteins.

The molecules mentioned in section 2) above are non-natural proteins.

In certain embodiments, the protein to be purified is an antibody.

The term "antibody" as used herein refers to an intact antibody or a binding fragment thereof that is equivalent to an intact antibody for specific binding. Binding fragments include, but are not limited to, single domain antibodies and single chain antibodies such as F(ab), F(ab'), F(ab') ₂ , Fv, VHH antibodies (nanobodies). The term "heavy chain" includes any immunoglobulin polypeptide that has sufficient variable region sequence to confer specificity for an antigen.

The term "heavy chain" as used herein includes full-length heavy chains and fragments thereof. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is located at the amino terminus of the polypeptide and the CH3 domain is located at the carboxyl terminus.

The term "light chain" as used herein includes full-length light chains and fragments thereof. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. Like the heavy chain, the variable region domain of the light chain is located at the amino terminus of the polypeptide. The term "light chain" as used herein includes any immunoglobulin polypeptide that has sufficient variable region sequence to confer specificity for an antigen.

Naturally occurring antibody structural units typically include tetramers. Each such tetramer typically consists of two homologous pairs of polypeptide chains, each pair consisting of one full-length light chain (typically having a molecular weight of about 25 kDa) and one It has a full-length heavy chain (typically having a molecular weight of about 50-70 kDa). The amino-terminal portion of each light and heavy chain typically contains a variable region of about 100-110 or more amino acids, which is typically involved in antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses, including, but not limited to, IgA1 and IgA2. In full-length light and heavy chains, typically the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain is joined by a "J" region of about 10 or more amino acids. D” area.

The variable region of each light/heavy chain pair typically forms an antigen binding site. The variable regions typically exhibit the same general structure with relatively conserved framework regions (FRs) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of the two chains of each pair are typically aligned by framework regions, which allow binding to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chain variable regions typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Amino acid assignments to each domain typically follow the definitions in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242. Bispecific or bifunctional antibodies are typically artificial hybrid antibodies that have two different heavy/light chain pairs and two different binding sites.

The F(ab) fragment consists of one light chain and CH1 and one heavy chain variable region. The heavy chain of an F(ab) molecule cannot form disulfide bonds with another heavy chain molecule. The F(ab') fragment contains one light chain and one heavy chain, which contains several constant regions between the CH1 and CH2 domains, thereby providing an interchain link between the two heavy chains. Disulfide bonds may be formed to form F(ab') ₂ molecules. The Fv region contains both heavy and light chain variable regions, but lacks constant regions. Single chain antibodies are Fv molecules in which the heavy and light chain variable regions are joined by a flexible linker to form a single polypeptide chain that forms the antigen binding region. It is understood that bivalent antibodies, other than "multispecific" or "multifunctional" antibodies, in certain embodiments, contain binding sites with the same antigen specificity.

Monoclonal antibodies (mAbs) that can be purified by the methods of the present disclosure can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as standard somatic cell hybridization techniques well known in the art. include. Although somatic cell hybridization techniques are preferred, other techniques for producing monoclonal antibodies can in principle be utilized, such as viral or oncogenic transformation of B lymphocytes. Monoclonal antibodies may correspond to, for example, murine, chimeric, humanized or fully human antibodies.

In addition, non-limiting examples of antibodies that can be purified by the method of the present invention include panitumumab, omalizumab, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, aftuzumab, alacizumab ), alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, apolizumab, atinumab, tocilizumab, basilizimab, bectumomab, belimumab, Bacizumab, bicilomab, canakinumab, cetuximab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, efungumab, ertumaxomab, etaracizumab, etanercept, golimumab, infliximab, natalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab, Rituximab, tocilizumab, trastuzumab, dupilumab, sarilumab or fresolimumab.

In certain embodiments, the protein to be purified is an enzyme.

Non-limiting examples of enzymes that can be purified by the methods of the invention include acid α-glucosidase, α-L-iduronidase, iduronic acid sulfatase, heparan N-sulfatase, galactose-6-sulfatase, acid β-galactosidase, β - Glucuronidase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylgalactosaminidase (α-galactosidase B), acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, β-glucosidase, galactosylceramidase, α-galactosidase A, acid β-galactosidase, β-galactosidase, neuraminidase, hexosaminidase A or hexosaminidase B.

Other non-limiting examples of proteins that may be purified by the methods of the invention include human erythropoietin, tumor necrosis factor (eg, TNF-α, TNF-β or TNF-K), interferon alpha or interferon beta.

The solution containing the protein to be purified can be a culture medium, preferably a clarified culture medium. The solution containing the protein to be purified is, for example, a culture medium obtained in a perfusion bioreactor or a fed-batch bioreactor.

Examples of perfusion bioreactors or fed-batch bioreactors are U.S. Patent Application Publication No. 2014/255994, U.S. Patent Application Publication No. 2015/232505, U.S. Patent Application Publication No. 2015/183821 and U.S. Patent Application Publication No. 2017/218012. and WO 2014/137903 (incorporated herein by reference in its entirety).

The term "clarified culture medium" means substantially free (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99%) of mammalian, bacterial or yeast cells. ) means a liquid culture medium obtained from mammalian, bacterial or yeast cell cultures.

In certain embodiments, the first filtration step of the method of the invention is integrated into a clarification step used to obtain a clarified culture medium in the recovery of cell cultures, whereby said filtration step can be part of the process.

The phrase "protein recovery" as used herein refers to harvesting the protein after use of the disclosed purification methods.

In the context of the present invention, the expression "chelating agent" refers to any kind of particulate adsorption medium or immobilized ligand, such as activated carbon, diatomaceous earth, bead resin, which acts as an absorbent in the purification process. The contaminant molecules present in the mixture are separated from the target molecules to be purified. The expression "chelating agent matrix" does not include Protein A matrix.

In certain embodiments of the methods of the invention, at least one chelating agent matrix is a particulate adsorption medium.

The at least one chelating agent matrix can be in the form of a column, filter, or added as a powder to the product to be purified. In certain embodiments, at least one chelating agent matrix used in the context of the present invention is added to the product to be purified as a powder.

In certain embodiments of the disclosed methods, at least one chelating agent matrix is an activated carbon filter. In certain other embodiments of the disclosed methods, the at least one chelating agent is a resin.

At least one chelating agent matrix, in particular an activated carbon filter or a resin medium, interacts with the contaminants, resulting in highly efficient removal of the impurities. Another advantage of using chelating agent matrices, particularly activated carbon or resins, is the low affinity for monoclonal antibodies.

In certain embodiments of the invention, the at least one chelating agent matrix of the filtration step is selected from the group consisting of activated carbon, diatomaceous earth, free cation exchange resins, free anion exchange resins, and free mixed mode resins. be done. In certain embodiments, the at least one chelating agent matrix is a combination of two different chelating agent matrices. In a more particular embodiment, the combination of two different chelating agent matrices is a combination of activated carbon, particularly an activated carbon filter, and a free anion exchange resin.

In certain embodiments of the disclosed methods, the activated carbon filter is Zeta Plus 35SP (commercialized by 3M), Zeta Plus 53SP (commercialized by 3M), Millistak CR40 (commercialized by Millipore), or Zeta Plus 55SP grade (commercialized by 3M Company).

In another specific embodiment of the disclosed method, the free anion exchange resin is NH2-750F (commercialized by Tosoh Corporation) or Emphaze AEX (commercialized by 3M Corporation). The properties of the resin NH2-750F are summarized below.

In the context of the present invention, the expression "diafiltration" refers to a technique that uses an ultrafiltration membrane to completely remove, replace or reduce the concentration of salts or solvents from a solution.

As used herein, "ultrafiltration" or "UF" refers to the use of semipermeable membranes to physically and selectively remove particles and/or ions from a solution based on the particle size and pore size of the UF membrane. refers to filtration technology that removes

In one embodiment, at least one diafiltration membrane of the exchange step is a single path tangential flow filtration (SPTFF) module or a tangential flow filtration (TFF) module. In certain embodiments, at least one diafiltration membrane of the exchange step is in the form of a cassette, hollow fiber or spiral.

In certain embodiments of the disclosed methods, at least one diafiltration membrane of the exchange step is a Ready To Process Hollow Fiber Cartridge 30 kDa (commercialized by GE).

In certain embodiments of the disclosed methods, at least one diafiltration membrane of the exchange step is a Cadence Inline Diafiltration (commercialized by Pall Inc.).

In one embodiment, the at least one diafiltration membrane of the exchange step is preceded or followed by a Cadence Inline Concentrator (commercialized by Pall Inc.).

In another specific embodiment of the disclosed method, the at least one diafiltration membrane of the exchange step is a Pellicon cassette (commercialized by Millipore) or a Sartocon cassette (commercialized by Sartorius).

The exchange step advantageously allows for both purification and concentration of the filtered protein solution. By the expression "concentration of a filtered protein solution" it is meant herein that the concentration of the protein in the partially purified retentate is increased compared to this concentration in the filtered protein solution. .

In the context of the present invention, "membrane adsorbent" refers to flat sheets of polymers, in particular acrylic polymers, or fibrous or non-woven media, having functional groups such as affinity groups and ion exchange groups. One of the differences between resin and membrane is the flow rate distribution due to diffusion through the resin and convection through the membrane.

The combination of membrane adsorbents used in the polishing process involves the use of at least two types of membrane adsorbents that are orthogonal in terms of mechanism of action.

In the context of the present invention, the expression "membrane adsorbents that are orthogonal with respect to their mode of action" means that the membrane adsorbents used have opposite or different mechanisms of action, e.g. cation exchange and anion exchange interactions, multimodal and anion exchange interactions, cation exchange and hydrophobic interactions, or anion exchange and hydrophobic interactions and process them together as if they were a single filtration step, integrated into one It means that it acts as a process.

In fact, the inventors have shown that the use of such a combination of membrane adsorbents makes it possible to capture impurities and, moreover, to minimize the adsorption of the desired product.

In preferred embodiments, where at least one additional membrane adsorbent is used for three or more membrane adsorbents in the polishing step membrane adsorbent combination, i.e. for a total of three, four or five or more membrane adsorbents, the The at least one additional membrane adsorbent is a membrane with a different mode of action compared to the two membrane adsorbents that are orthogonal with respect to the mode of action, and this total of three or more membrane adsorbents is one It is still considered to be a single process integrated with the

As a non-limiting example, said at least one additional membrane adsorbent may be hydrophobic if the two membrane adsorbents, which are orthogonal with respect to mechanism of action, have cation exchange and anion exchange interactions, respectively. Has interaction or polymorphic interaction.

In the context of the present invention, membrane adsorbents are in particular combined as one integrated process. Therefore, the number of steps and buffers is significantly reduced. Also, adjustments and manipulations are eliminated, thus simplifying the overall method. In particular, there are no elution, conditioning or storage steps between one membrane adsorbent of a combination of membrane adsorbents and the other membrane adsorbent(s) of such a combination of membrane adsorbents.

In certain embodiments, the particular combination of membrane adsorbents and operating conditions (particularly pH, conductivity and/or buffers) of the polishing step are determined according to the physicochemical properties (e.g. pI, molecular weight, etc.) of the protein to be purified. It is determined.

As will be understood by those skilled in the art, optimal conditions for the polishing step make it possible to obtain the maximum yield of the protein of interest, i.e., the lowest interaction of the protein of interest with the membrane adsorbent combination. Furthermore, maximum contaminant removal should be possible, i.e. without the need for any conditioning or elution steps between the membrane adsorbent combination and the contaminant removal process. The highest possible interaction of objects should be possible.

An example of determining the optimal combination of two membrane adsorbents for a polishing process is illustrated in FIG. In this example, the best purification capacity is obtained with high contaminant removal rates (low HCP and HMW) while maximizing antibody yield using a combination of Sartobind S and Sartobind STIC membrane adsorption. The effect of loading capacity on yield and contaminant removal is also shown in this figure.

In the context of the present invention, in order to determine these optimal conditions, the membrane adsorbents of the membrane adsorbent combination are combined into a single unit or one, processing them together as if in a single filtration step. It should be considered an integrated process. For example, in the case of two membrane adsorbents, when using two membrane adsorbents, the traditional method of determining optimal conditions is to determine the best conditions for the first membrane adsorbent, then In the context of the present invention, the two membrane adsorbents is considered to be unique, and a compromise solution should be determined if the separation behavior of the two membrane adsorbents still makes it possible to achieve a good purification.

Typically, when determining the optimized buffer to use for the purification of a given protein, the exchange step can be performed with a neutral buffer, and the pH and conductivity of the solution containing the residual liquid can be manually determined. can be adjusted to determine the optimal conditions for purification in the polishing process. When determining optimal conditions, the method can be run again using a suitable buffer for the exchange step, which corresponds to the equilibration buffer used for the polishing step.

An example of determining the optimal buffer (eg, buffer conductivity or pH) and pI of the protein of interest by a combination of two membrane adsorbents used in the polishing step is illustrated in FIG. In this example, the pH and conductivity conditions that make it possible to achieve advantageous purification (corresponding to HCP levels of 50-500 ng/ml) correspond to the black areas in each plot.

In the context of the present invention, one buffer used for equilibration of at least two membrane adsorbents is the same as the buffer used for the exchange step. This allows direct flow-through of the protein to be purified, maximizing yield while retaining most of the contaminants.

In certain embodiments, the polishing step is optimized by varying the pH and conductivity of the buffer used to condition the protein during the exchange step.

In certain embodiments, the polishing step includes the use of a cation exchange membrane adsorption matrix in combination with an anion exchange membrane adsorption matrix. In other embodiments, the polishing step includes the use of a multimodal (mixed mode) membrane adsorption matrix in combination with an anion exchange membrane adsorption matrix.

Combinations of membrane adsorption matrices, particularly cation-exchange membrane adsorbents and anion-exchange membrane adsorbents, function through interactions between the membrane adsorbents and contaminants to remove impurities with high efficiency. Interactions with contaminants are due to several mechanisms: ionic, hydrophobic, van der Waals forces and hydrogen bonding interactions.

In certain embodiments, the cation exchange membrane adsorbent is a Sartobind S membrane adsorbent (Sartorius), an HD-C membrane adsorbent (Natrix). In certain embodiments, the cation exchange membrane adsorbent is a Sartobind S membrane adsorbent (Sartorius). In certain embodiments, the anion exchange membrane sorbent is a Sartobind STIC membrane sorbent (Sartorius), a Sartobind Q membrane sorbent (Sartorius), or a HD-Q membrane sorbent (Natrix). In certain embodiments, the anion exchange membrane adsorbent is a Sartobind STIC membrane adsorbent (Sartorius) or a Sartobind Q membrane adsorbent (Sartorius). In other embodiments, the multimodal membrane adsorbent is an HD-Sb membrane adsorbent (Natrix). In other embodiments, the hydrophobic interaction membrane adsorbent is Sartobind Phenyl membrane adsorbent (Sartorius).

In certain embodiments, the membrane adsorbent combination is a combination of Sartobind S membrane adsorbent (Sartorius) and Sartobind STIC membrane adsorbent (Sartorius).

The main advantages of using a membrane adsorbent rather than a column in the polishing step of the method of the invention are summarized below.
- On a comparable scale, membrane adsorbents can be used at flow rates 10 times higher than columns, which significantly reduces processing times. For example, a 5 mL column packed with resin is used at a flow rate of 1 ml/min, while a corresponding 5 mL membrane adsorbent is used at a minimum flow rate of 10 ml/min. As a result, traditional methods that use two chromatographic steps for polishing, performed for 2 hours and 30 minutes using two resin-filled columns, can be completed in less than 15 minutes using the membrane adsorbent. I can do it.
- Although the column is reusable, the membrane adsorbent is a disposable device, so it can be discarded after the batch and does not require long-term storage. Therefore, there is no need to test them to ensure long-term stability.
- Using membrane adsorbents is cheaper by avoiding column costs, column packing and column storage.

In certain embodiments, when the combination of membrane adsorbents is a combination of Sartobind S membrane adsorbent (Sartorius) and Startobind STIC membrane adsorbent (Sartorius), one buffer is and have a pH and conductivity in the range disclosed in the area.

In another specific embodiment, when the membrane adsorbent combination is a combination of Sartobind S membrane adsorbent (Sartorius) and Startbind Q membrane adsorbent (Sartorius), one buffer is It has a pH and conductivity in the range disclosed in the black area.

The purification method according to the invention is a complete flow-through purification method.

A "complete flow-through purification method" is meant herein to include the meaning that all of the various purification steps of the method involve impurities binding only while the protein of interest is being passed through the purification steps.

In one embodiment, the method according to the invention does not involve adjusting the pH of the filtered protein solution at the end of the filtration step and/or of the retentate at the end of the exchange step.

In certain embodiments, the filtered protein solution obtained at the end of the filtration step is passed directly through a diafiltration membrane. More particularly, no treatment (eg pH adjustment, buffer exchange or dilution) is then performed between the two steps. In such methods, the diafiltration membrane may correspond to, for example, a Ready To Process 30 kD hollow fiber module or a Cadence Inline Diafiltration module, preceding or following, for example, a Cadence Inline Concentrator. Additionally, in certain embodiments, the residual liquid obtained at the end of the exchange step is passed directly through the membrane adsorbent combination of the polishing step. More particularly, no treatment (eg pH adjustment, buffer exchange or dilution) is then performed between the two steps. In such a method, the diafiltration membrane is comprised of a combination of membrane adsorbents, which may correspond, for example, to a Ready To Process 30kD hollow fiber module, and/or a combination of Sartobind S membrane adsorbents and Sartobind STIC membrane adsorbents, for example. I can handle it.

In such methods, there are no intermediate steps that require manual intervention and opening of the purification system (eg dilution, conductivity adjustment and pH adjustment).

Thus, the method of the invention can be carried out in a flexible, automated chromatography system for operation in a series or sequential three purification steps, including multiple pumps and sensors with wiring and switching valves.

A non-limiting example of a multiplex operating system is a suitably adapted multiple column chromatography system MCCS.

The method of the invention can be carried out in a continuous manner. In other words, the method of the invention can be a continuous method for purifying proteins from solution.

The term "continuous process" or "continuous process" refers to a process in which liquid is continuously supplied through at least a portion of the system.

By the term "liquid" is meant herein any liquid, such as a solution containing the protein to be purified, a buffer or a solution of low or acidic pH for virus inactivation.

In certain embodiments, the first, second, and third purification steps deliver liquid sequentially.

The term "integrated method" refers to a method performed using structural elements that work cooperatively to achieve a specific result, such as the production of purified protein from a liquid culture medium.

Furthermore, the method of the invention can be carried out in a closed system from the first step to the last step of the method. In other words, the method of the invention preferably has a functionally closed channel. In particular, the three purification steps and optionally the filtration step(s) (e.g. the nanofiltration step and/or the final ultrafiltration and/or diafiltration step) can be carried out in a closed system. . In a particular embodiment of the method of the invention, a solution containing the protein is passed in portions through three purification steps, each passage of a portion of the solution corresponding to a run. The protein recovered at the end of each run is then harvested and stored. In such methods, the membrane adsorbent of the purification process is used several times and is optionally sanitized, e.g. using a sanitizing buffer as defined above, so that the membrane adsorption device and the required buffer are The amount can be reduced. For example, a series of 3-50 runs (eg, 3-30, 5-25, 10-20, or 15 runs) can be performed consecutively. More particularly, after 3, 4, 5, 6, 7 or 8 runs are performed in a sequential manner, the membrane adsorbent can be sanitized (eg, using a sanitizing buffer). This may be repeated, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, as illustrated in FIG.

Purified proteins can be recovered using the methods disclosed herein. As used herein, "purified" refers to a purity that allows for effective use of the protein in vitro, ex vivo, or in vivo. Proteins that are useful in in vitro, ex vivo, or in vivo applications are free from contaminants, other proteins, and/or inclusions in the protein of interest that may interfere with the use of the protein in such applications. should be at least substantially free of undesirable chemicals. Such applications include the preparation of therapeutic compositions, administration of proteins in therapeutic compositions, and other methods disclosed herein. Preferably, a "purified" protein as referred to herein can be produced by any method (i.e., by direct purification from a natural source, recombinantly, or synthetically) and the protein is , in a given composition, at least about 70% weight/total protein weight, more preferably at least about 80%, or at least about 85%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably is purified from other protein components to contain at least about 97%, more preferably at least about 98%, more preferably at least about 99% weight/weight of total protein in a given composition. It is a protein.

In certain embodiments, the method of the invention comprises:
(a) a filtration step,
(i) passing the solution through the at least one chelating agent matrix in a flow-through manner;
(ii) recovering a filtered protein solution from the flow-through of the at least one chelating agent matrix;
(b) a replacement step,
(i) passing the filtered protein solution obtained from step (a) and one buffer through at least one diafiltration membrane;
(ii) recovering a residual liquid of the at least one diafiltration membrane comprising partially purified protein; and (c) a polishing step, the step comprising: (i) adding an equilibration buffer to the membrane. passing through a combination of adsorbents, said equilibration buffer being the same as one buffer used for exchange in step (b);
(ii) passing the residual liquid obtained from step (b) through the membrane adsorbent combination in a flow-through manner;
(iii) recovering purified protein from the flow-through of the membrane adsorbent combination, wherein the purification method does not include a Protein A chromatography step;
One buffer is
- 5-40mM (e.g. 20mM) Bistris and 15-150mM (e.g. 75mM) NaCl, adjusted to a pH of 6-9 (e.g. 7.5) with acetic acid;
- 5-40mM (e.g. 20mM) Tris or Tris-HCl, 15-150mM (e.g. 75mM) NaCl, adjusted to a pH of 6-9 (e.g. 7.5) with acetic acid;
- 5-40mM (e.g. 20mM) Tris or Tris-HCl, 15-150mM (e.g. 75mM) NaCl, adjusted to a pH of 6-9 (e.g. 7.5) with citric _acid , or - _Na2HPO4 / _NaH2PO4 (preferably from 10mM/90mM _{Na2HPO4 / NaH2PO4} to 90mM/ _{10mM Na2HPO4} / _NaH2PO4 ) to a pH _of 6-9 (eg _7.5 ) _. Adjusted 15-150mM (e.g. 75mM) NaCl
Contains or consists of these.

The method for purifying proteins from solution may include at least one additional final filtration step, such as a nanofiltration step, an ultrafiltration step and/or a diafiltration step, after the polishing step. When purifying recombinant proteins for pharmaceutical purposes, a polishing step is typically followed by an additional final filtration step. Therefore, the method of the invention may further include a nanofiltration step after step (c). Ultrafiltration and diafiltration steps may further be performed after the nanofiltration step. As used herein, "ultrafiltration" or "UF" refers to the use of semipermeable membranes to physically and selectively remove particles and/or ions from a solution based on the particle size and pore size of the UF membrane. refers to filtration technology that removes As used herein, "nanofiltration" refers to the filtration of a solution through a nanofilter used, for example, to remove viral particles. As used herein, "diafiltration" refers to a technique that uses an ultrafiltration membrane to completely remove, replace, or reduce the concentration of salt or solvent from a solution.

The method of the invention may also further include at least one virus inactivation step. Said at least one virus inactivation step can be carried out at any stage of the method of the invention, for example before step (a), after step (a), after step (b), after step (c). It may take place after a filtration step and/or after an ultrafiltration and/or diafiltration step. Such virus inactivation steps can typically be low pH or acidic pH inactivation steps. As used herein, "low pH or acidic pH inactivation" refers to virus inactivation techniques that use acidic pH to denature viruses, particularly enveloped viruses. Typically, the low pH or acidic pH inactivation step is about 3.0 to 5.0 (eg, about 3.5 to about 4.5, about 3.5 to about 4.25, about 3.5 to about 4.0, such as 4.0) for a period of at least 15 minutes (e.g., a period of 15 minutes to 1 hour, a period of about 30 minutes to 2 hours, or a period of about 45 minutes to 2 hours) , by incubating the recovered proteins. For example, a low pH or acidic pH inactivation step is performed by incubating the recovered protein at pH 4 for, eg, 30 minutes to 2 hours.

The method of the present invention may also include, before step (a), providing a liquid culture medium containing the protein to be purified that has been clarified to remove cells and is substantially free of cells. Yes, in which case the liquid culture medium is fed through at least one chelating agent matrix.

For example, the method of the invention for purifying proteins from solution can include
(before a) preparing a liquid culture medium containing the protein to be purified that has been clarified to remove cells and is substantially free of cells;
(a) a filtration step,
- passing said liquid culture medium of step (a) in a flow-through manner through at least one chelating agent matrix;
- recovering a filtered protein solution from the flow-through of said at least one chelating agent matrix;
(b) a replacement step,
- passing the filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for exchange;
- recovering a retentate of said at least one diafiltration membrane comprising partially purified protein; and (c) a polishing step, comprising:
- passing the residual liquid obtained at the end of step (b) in a flow-through manner through a combination of membrane adsorbents, the two membrane adsorbents of said combination of membrane adsorbents having a mechanism of action; and the combination of membrane adsorbents is pre-equilibrated with an equilibration buffer identical to the one buffer used for exchange in step (b);
- recovering the purified protein from the flow-through of said combination of membrane adsorbents, the purification method not including a chromatography step with Protein A.

Ultimately, the purified protein may be formulated into a composition suitable for storage and/or in particular a pharmaceutical composition suitable for administration to animals and/or humans.

One of the many advantages of the disclosed method is that it makes it possible to obtain good yields of highly pure proteins. The purified protein recovered by the method of the invention can be, for example, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2% , 99.5% or 99.9% purity. More particularly, one of the many advantages of the methods of the present disclosure is the reduction in the amount of contaminating DNA, high molecular weight (HMW) species (corresponding to protein aggregates) and/or host cell proteins (HCP). It is now possible to obtain highly pure protein solutions containing A solution containing purified protein recovered by the method of the invention may exhibit an amount of contaminant DNA of, for example, less than 0.4 ppb, less than 0.3 ppb, less than 0.2 ppb, or less than 0.1 ppb. Further, the solution containing purified protein recovered by the method of the present invention may be, for example, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or Concentrations of HMW species of less than 0.1% may be indicated. In addition, the solution containing purified protein recovered by the method of the present invention may be, for example, less than 500 ng/ml, less than 100 ng/ml, less than 90 ng/ml, less than 85 ng/ml, less than 80 ng/ml, less than 75 ng/ml, or A concentration of HCP of less than 70 ng/ml may be indicated. In addition, the methods of the invention allow recovery of purified protein in a yield of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. shall be.

The present invention further includes:
(a) a combination of at least one chelating agent matrix, at least one diafiltration membrane, and a membrane adsorbent, wherein the two membrane adsorbents of the membrane adsorbent combination are orthogonal with respect to mechanism of action; a combination of membrane adsorbents and (b) Tris, Tris-HCl, Bis-Tris, phosphoric acid and/or citric acid, in particular (i) Bis-Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) a buffer comprising or consisting of water, and (iv) optionally NaCl, and/or Tris, Tris-HCl, Bis-Tris, phosphoric acid and/or citric acid, in particular (i) Bis-Tris, Contains or consists of instructions for preparing one buffer containing or consisting of Tris or Tris-HCl, (ii) acetic acid, (iii) water, and (iv) optionally NaCl. , regarding the kit.

The invention is further illustrated by the following figures and examples.

FIG. 1 is a schematic diagram representing the three-step complete flow-through method of the present invention. FIG. 4 is a schematic representation of a continuous version of the three-step complete flow-through method of the present invention used in Example 4. Membrane yield (%), HMW (%) and HCP (ng/ml) obtained by four combinations of membrane adsorbents (HD-C+HD-Q, HD-Sb+HD-Q, Sartobind S+Q, Sartobind S+STIC) It is a histogram showing a comparison by volume (mg/ml). Purification of two combinations of membrane adsorbents (top panel: Sartobind S+STIC, bottom panel: Sartobind S+Q) and three pIs (6; 7.5 and 9) by pH and conductivity (mS/cm) of the equilibration buffer. FIG. 2 is a plot of the sweet spot for proteins that The pH and conductivity conditions that make it possible to achieve advantageous purification (corresponding to HCP levels of 50-500 ng/ml) correspond to the black area in each plot. Partial chromatogram of purification through membrane (UV 280 nm) in a laboratory scale continuous method experiment.

Laboratory-scale method according to the invention The method of the invention was utilized for laboratory-scale batch purification of humanized monoclonal antibody mAb1.

This experiment shows the impurity removal results achieved on a laboratory scale after three steps. The goal was to remove aggregates (HMW) and host cell proteins (HCP) to less than 1% for HMW and less than 500 ng/ml for HCP.

The inventors have successfully used two different chelating agents, one exchange step and a pair of membranes (Sartobind S and STIC or Sartobind S and Q).

Laboratory-scale method according to the invention The method of the invention was utilized for laboratory-scale batch purification of humanized monoclonal antibody mAb2.

This experiment shows the impurity removal results achieved on a laboratory scale after three steps. The goal was to remove aggregates (HMW) and host cell proteins (HCP) to less than 1% for HMW and less than 500 ng/ml for HCP. The inventors have successfully used two chelating agents, one exchange step and a pair of membranes (Sartobind S and STIC or Sartobind S and Q). The best results were obtained using a pair of Sartobind S+STICs with loading conditions set at pH 8.5 and 3 ms/cm.

Pilot-scale method according to the invention The method of the invention was utilized for pilot-scale batch purification of mAb1.

The same method disclosed in Example 2 was scaled up to pilot scale. The goal was to purify 10 g through a nanofiltration step after three steps.

8 L of clarified cell culture supernatant was purified through three steps of the method of the invention and nanofiltration. 8L is 250 ml of Tosoh NH ₂ -750F resin, then stirred for 13 minutes, 160 g of activated carbon (Norit SA2 grade), then stirred for 14 minutes, 30 g of activated carbon (Norit SA2 grade), then stirred for 10 minutes. Continuously introduced into a 20 L stirrer.

After filtering the product (Filtrox filter), the exchange process was started. Approximately 9 L of product was recovered after filtration (the product was forced out of the filter with 1 L of sterile water). UF/DF was performed on a GE Uniflux using a hollow fiber (790 cm ² -50KD-Spectrumlab). The buffer used for diafiltration of the product through hollow fibers was adjusted to pH 7.2 with acetic acid. S. of 20mM Bis-Tris buffer.

2.64 kg was recovered at 6.9 g/L (18.2 g of monoclonal antibody). The 2 L (14 g) of recovered product was then extruded through two membrane adsorbents: Sartobind S for 75 ml and Sartobind Q for 200 ml. The two membranes were coupled sequentially and used in a flow-through mode using the GE AktaProcess.

Finally, the product was nanofiltered through a prefilter (XOHC-Millipore) and a Viresolve Pro filter (Millipore) to obtain the final quality.

The table below shows a comparison between the conventional method and the method of the invention.

Laboratory-scale fully continuous method according to the invention The method of the invention was utilized for laboratory-scale batch purification of humanized monoclonal antibody mAb1 in a continuous manner.

The goal of the experiment was to purify mAb1 from the clarified bulk harvest using a continuous mode, meaning no interruptions, storage, or adjustments between each step. The inventors successfully used filtration over a chelating agent (immobilized AEX, activated carbon CA), one exchange step (single pass diafiltration) and a pair of membranes (Sartobind S and STIC). did.

3L of clarified cell culture supernatant was purified through three steps of the method of the invention. The product was filtered through an immobilized anion exchanger (Emphaze AEX BV120) and then through an activated carbon filter (Millipore CR40 270 cm2). The product was then passed directly through a single pass diafiltration membrane (0.2 m2) for concentration and diafiltration (exchange step). The buffer used for diafiltration through the product was adjusted to pH 7.5 with acetic acid. S. 20mM Bis-Tris, 20mM NaCl buffer.

The diafiltration product was collected directly from the retentate side, which was passed through an intermediate surge bag, to accommodate flow rate differences due to later steps. The product was then further extruded through two polishing membrane adsorbents: Sartobind S for 1 ml and Sartobind STIC for 1 ml. The two membranes were coupled sequentially and used in a flow-through format using GE Akta Pure. Each unit process was either directly connected to the other or processed sequentially through a surge bag.

Multiple cycles of purification were performed on a membrane adsorbent to process the entire volume of product as shown in Figure 5 (excerpt from 50 membrane purification cycles). The entire product pool was collected through a sterile filter at the end of the process. Purification of 40 mg of mAb every 5 minutes resulted in a productivity of 240 g of mAb1 per liter of membrane per hour (240 g/L/h).

Claims (20)

A method for purifying a protein from a solution, the method comprising:
(a) a filtration step,
- passing the solution through at least one chelating agent matrix in a flow-through manner;
- recovering a filtered protein solution from the flow-through of at least one said chelating agent matrix;
(b) a replacement step,
- passing the filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for exchange;
- recovering a retentate of at least one said diafiltration membrane comprising partially purified protein;
(c) a polishing step,
- passing the residual liquid obtained at the end of step (b) in a flow-through manner through a combination of membrane adsorbents, the two membrane adsorbents of said combination of membrane adsorbents having a mechanism of action; and the combination of membrane adsorbents is pre-equilibrated with an equilibration buffer identical to the one buffer used for exchange in step (b);
- recovering the purified protein from the flow-through of said combination of membrane adsorbents, not including a chromatography step with Protein A, wherein the membrane adsorbent of the polishing step is a cation exchange membrane adsorbent; and an anion exchange membrane adsorbent, the protein being an antibody . 4. The filtered protein solution obtained at the end of step (a) is directly passed through at least one diafiltration membrane without undergoing any treatment such as pH adjustment, buffer exchange or dilution. The method described in 1. 2. The residual liquid obtained at the end of step (b) passes directly through the membrane adsorbent combination without undergoing any treatment such as pH adjustment, buffer exchange or dilution. The method described in. A method according to any one of claims 1 to 3, which does not include any intermediate storage between the three steps (a), (b) and (c). A method according to any one of claims 1 to 4, having a functionally closed channel. A method according to any one of claims 1 to 5, wherein only one buffer is used throughout the purification method. One buffer includes Tris, Tris-HCl, Bis-Tris, phosphoric acid and/or citric acid, particularly (i) Bis-Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) water, and (iv) A method according to any one of claims 1 to 6, optionally comprising or consisting of a salt. 8. The method of claims 1-7, wherein the at least one chelating agent matrix of the filtration step is selected from the group consisting of activated carbon, diatomaceous earth, free cation exchange resins, free anion exchange resins, and free mixed mode resins. The method described in any one of the above. 9. The method of claim 8, wherein the at least one chelating agent matrix is a combination of two different chelating agent matrices. 10. The method of claim 9, wherein the combination of two different chelating agent matrices is a combination of activated carbon and free anion exchange resin. A method according to any one of the preceding claims, wherein at least one diafiltration membrane of the exchange step is a single path tangential flow filtration (SPTFF) module or a tangential flow filtration (TFF) module. A method according to any one of claims 1 to 11, wherein at least one diafiltration membrane of the exchange step is in the form of a cassette, hollow fiber or spiral. A method according to any one of claims 1 to 12, wherein the filtered protein solution is concentrated during the exchange step. The method according to any one of claims 1 to 13 , wherein the combination of membrane adsorbents in the polishing step is a combination of a cation exchange membrane adsorbent and an anion exchange membrane adsorbent. The method according to any one of claims 1 to 14 , further comprising a nanofiltration step after step (c). 16. The method of claim 15 , further comprising a final ultrafiltration and/or diafiltration step after the nanofiltration step. 17. The method according to any one of claims 1 to 16 , wherein the protein is a monoclonal antibody. One buffer is
- 5-40mM Bistris, 15-150mM NaCl, adjusted to pH 6-9 with acetic acid;
- 5-40mM Tris or Tris-HCl, 15-150mM NaCl, adjusted to a pH of 6-9 with acetic acid;
- adjusted to a pH of 6 to 9 with citric acid, 5 to 40 mM Tris or Tris-HCl, 15 to 150 mM NaCl, or - adjusted to a pH of 6 to 9 with Na ₂ HPO ₄ /NaH ₂ PO ₄ , 15-150mM NaCl
18. The method according to any one of claims 1 to 17 , comprising: 19. The method according to any one of claims 1 to 18 , further comprising the step of formulating the recovered purified protein into a pharmaceutical composition. (a) a combination of at least one chelating agent matrix, at least one diafiltration membrane, and a membrane adsorbent, wherein the two membrane adsorbents of said membrane adsorbent combination are orthogonal with respect to their mechanism of action; , a combination of membrane adsorbents, including a cation exchange membrane adsorbent and an anion exchange membrane adsorbent , and (b) one buffer comprising Tris, Tris-HCl, Bistris, phosphoric acid and/or citric acid. or a kit suitable for purifying proteins from a solution according to the method according to any one of claims 1 to 19 .

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